Reproduction in flowering plants involves two fertilization events. A sperm fuses with the egg cell to form a zygote which becomes the embryo; a second sperm cell fuses with the doubled-haploid central cell nucleus to form the starting point of the triploid endosperm tissue. While fertilization is thus normally the trigger for seed development, mutants have been identified in which reproductive processes are initiated independent of fertilization. Such mutations uncouple components of seed development from the fertilization process, resulting in developmental patterns resembling those found in apomictic plants.
Arabidopsis fie mutants (for fertilization-independent endosperm) isolated by Ohad et al. (Proc. Natl. Acad. Sci. USA 93:5319-5324, 1996; see also U.S. Pat. No. 6,229,064) exhibit replication of the central cell nucleus, initiating endosperm development, in the absence of fertilization. Inheritance of the mutant fie allele by the female gametophyte results in embryo abortion; thus, the trait can be transmitted to progeny only by the male gametophyte. The Arabidopsis FIE gene was cloned (Ohad et al., The Plant Cell 11:407-416 (1999); GenBank entry AF129516) and found to encode a polypeptide related to the WD Polycomb group proteins encoded by, for example, Esc in Drosophila (Gutjahr et al., EMBO J 14:4296-4306 (1995); Sathe and Harte, Mech. Dev. 52:77-87 (1995); Jones and Gelbart, Mol. Cell. Biol. 13:6357-6366 (1993). WD polycomb proteins may interact with other polynucleotides to form complexes which interfere with gene transcription (Pirrotta, Cell 93:333-336 (1998). Fertilization may trigger alteration of the protein complexes, allowing transcription of genes involved in endosperm development. Thus, loss-of-function fie mutants would lack the ability to form the protein complexes which repress transcription, and endosperm development could proceed independent of fertilization (Ohad et al. 1999, supra).
Chaudhury et al. (Proc. Natl. Acad. Sci. USA 94:4223 (1997)) reported fis (fertilization-independent seed) mutants in Arabidopsis. In fis1 and fis2 seed, the endosperm develops to the point of cellularization before atrophying. Proembryos are formed in a low proportion of seeds but do not develop beyond the globular stage. The FIS1 and FIS2 genes were cloned and further characterized. The FIS2 gene comprised structures suggesting function as a transcription factor; the FIS1 gene was found to be allelic (Proc. Natl. Acad. Sci. USA 96:296 (1999)) to the Arabidopsis gene MEDEA (Grossniklaus et al. Science 280:446 (1998)).
Apomixis (asexual reproduction) may occur through vegetative reproduction or through agamospermy, the formation of seeds without fertilization. Generally, agamospermy has not been exploited in agriculture; however, it has numerous potential applications, including perpetuation of high yielding crop plant hybrids and varieties, and maintenance of pure inbred lines. Also, seed formation without fertilization avoids factors that can reduce the efficiency of seed set, such as pollen count and pollen viability, and stigma or anther emergence or viability. Agamospermy would also allow the immediate stable incorporation of transgenes without the need for selfing to produce homozygotes. In addition, the fertilization-independent endosperm gene and other related genes could be used to cause the formation of a fertilization-independent endosperm without necessarily forming a viable embryo. Such a seed would not germinate because it lacks an embryo. However, the endosperm, if sufficiently formed, could be used for human and animal food and for commercial milling and extraction. Such embryo-less seeds would have the added advantage of allowing containment of genetically modified organisms to satisfy environmental and regulatory concerns. Such seeds could also be independently modified to produce novel products in the endosperm such as pharmaceuticals, nutraceuticals, and industrial compounds and polymers.
Identification of specific genes involved in agamospermy, such as fertilization-independent endosperm genes, will offer new ways of producing apomictic plants. Such approaches may involve selective mutagenesis of fertilization-independent endosperm genes and then tracking of the mutant alleles in a molecular breeding program, or transgenic methods. Accordingly, identification and isolation of nucleic acid sequences encoding all or a portion of a protein affecting seed development independent of fertilization would facilitate studies of developmental regulation in plants and provide genetic tools to engineer apomixis.